US10717999B2 - Method for the fermentative production of L-lysine using C. glutamicum strains with a mutated kup transporter - Google Patents

Method for the fermentative production of L-lysine using C. glutamicum strains with a mutated kup transporter Download PDF

Info

Publication number
US10717999B2
US10717999B2 US16/701,634 US201916701634A US10717999B2 US 10717999 B2 US10717999 B2 US 10717999B2 US 201916701634 A US201916701634 A US 201916701634A US 10717999 B2 US10717999 B2 US 10717999B2
Authority
US
United States
Prior art keywords
lysine
amino acid
seq
positions
bacterium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
US16/701,634
Other languages
English (en)
Other versions
US20200190546A1 (en
Inventor
Thomas BEKEL
Kornelia VOSS
Georg Thierbach
Frank Schneider
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evonik Operations GmbH
Original Assignee
Evonik Operations GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Evonik Operations GmbH filed Critical Evonik Operations GmbH
Assigned to EVONIK OPERATIONS GMBH reassignment EVONIK OPERATIONS GMBH ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SCHNEIDER, FRANK, BEKEL, Thomas, Voß, Kornelia, THIERBACH, GEORG
Publication of US20200190546A1 publication Critical patent/US20200190546A1/en
Application granted granted Critical
Publication of US10717999B2 publication Critical patent/US10717999B2/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02004—Aspartate kinase (2.7.2.4)

Definitions

  • the present application is accompanied by an ASCII text file as a computer readable form containing the sequence listing, titled “Sequence-Listing-as-filed.txt,” created on Tuesday, 15 Nov. 19, 2019, 11:22:59 AM, with the file size of 44,000 bytes, which is incorporated by reference in its entirety. Applicants hereby state that the information recorded in computer readable form is identical to the written (on paper or compact disc) sequence listing.
  • L-lysine is produced by fermentation of strains of the species Corynebacterium glutamicum . Work is continually being done on improving the production methods.
  • Improvements may relate to the fermentation technology, to the processing of the fermentation broth to a suitable product form or may relate to the intrinsic performance properties of the microorganism itself.
  • nucleotide sequences of the chromosomes of various bacteria strains of the species Corynebacterium glutamicum are available at publicly accessible databases and may be used for strain development purposes.
  • One such database is the GenBank database of the NCBI (National Center for Biotechnology Information. U.S. National Library of Medicine 8600 Rockville Pike, Bethesda Md., 20894 USA).
  • locus_tag a unique identifier
  • nucleotide sequence of the Corynebacterium glutamicum ATCC13032 chromosome and its analysis were described e.g. by Nakagawa et al. in EP1108790 A2.
  • the information is available at the NCBI under accession number NC_003450.
  • locus_tag NCgl0682 identifies a nucleotide sequence annotated as coding for a K+ transporter.
  • the amino acid sequence of the polypeptide is available under the identifier NP_599944.
  • the nucleotide sequence of the Corynebacterium glutamicum ATCC13032 chromosome and its analysis were independently described by Kalinowski et al. (Journal of Biotechnology 104 (1-3), 5-25 (2003)). The information is available at the NCBI under accession number NC_006958. Locus_tag CGTRNA_RS003565 identifies a nucleotide sequence annotated as coding for a potassium transporter Kup. The old_locus_tag designation cg0817 is also used in the art.
  • the amino acid sequence of the polypeptide is available under the identifier WP_011013837.
  • the nucleotide sequences of locus_tag NCgl0682 and CGTRNA_RS03565 are identical.
  • the amino acid sequence of the Kup transporter is shown as SEQ ID NO:2.
  • the corresponding nucleotide sequence of the kup gene is the gene identified by NCgl0682 and shown under SEQ ID NO: 1.
  • WO 01/00805 also discloses the kup gene of Corynebacterium glutamicum according to SEQ ID NO: 2 as well as other genes of C. glutamicum encoding proteins involved in membrane synthesis and membrane transport and generally teaches that modifying C. glutamicum strains in these genes increases the production of e.g. L-lysine.
  • Follmann et al. (Journal of Bacteriology 191(9), 2944-2952, 2009) investigated the potassium transport in Corynebacterium glutamicum . They provided experimental evidence that the potential potassium channel CglK is the only functional potassium uptake system in Corynebacterium glutamicum . Said potassium channel CglK is encoded by a nucleotide sequence identified by the old locus_tag designation cg0887. Follmann et al. further found that cells lacking the kup gene identified by old locus_tag cg0817 showed no difference in growth as compared to the wild type.
  • Object of the present invention is to provide new measures for the fermentative production of L-lysine by bacteria of the species Corynebacterium glutamicum.
  • a method for the fermentative production of L-lysine comprising the steps of providing a bacterium of the species Corynebacterium glutamicum having the ability to excrete L-lysine and containing in its chromosome a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the amino acid glycine at position 344 is substituted by valine, cultivating the bacterium in a suitable medium under suitable conditions, and accumulating L-lysine in the medium to form an L-lysine containing fermentation broth.
  • the polynucleotide encoding said amino acid sequence comprises the nucleotide sequence of positions 40 to 1923 of SEQ ID NO: 1 the nucleobases at positions 1069 to 1071 being gtt, gtc, gta or gtg.
  • the polynucleotide encoding said amino acid sequence comprises the nucleotide sequence of positions 40 to 1926 of SEQ ID NO: 1 the nucleobases at positions 1069 to 1071 being gtt, gtc, gta or gtg.
  • the polynucleotide encoding said amino acid sequence comprises the nucleotide sequence of SEQ ID NO: 1 the nucleobases at positions 1069 to 1071 being gtt, gtc, gta or gtg.
  • the bacterium contains at least one copy of a polynucleotide coding for a feedback resistant aspartate kinase polypeptide variant desensitized to inhibition by mixtures of L-lysine and L-threonine.
  • amino acid sequence of said feedback resistant aspartate kinase polypeptide comprises the amino acid sequence of SEQ ID NO:6 containing isoleucine instead of threonine at position 311.
  • the present invention makes available a novel method for the fermentative production of L-lysine comprising the steps of providing a bacterium of the species Corynebacterium glutamicum , having the ability to excrete L-lysine, containing in its chromosome a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2, wherein the amino acid glycine at position 344 is substituted by valine, cultivating the bacterium in a suitable medium under suitable conditions, and accumulating the L-lysine in the medium to form an L-lysine containing fermentation broth.
  • the L-lysine production is increased compared to a method comprising the cultivation of an L-lysine secreting bacterium of the species to Corynebacterium glutamicum without such substitution.
  • amino acid sequence of SEQ ID NO:2, wherein the amino acid glycine at position 344 is substituted by valine, is shown in SEQ ID NO:4.
  • modified bacteria provided in the method according to the invention excreted L-lysine, into a suitable medium under suitable fermentation conditions in an increased manner.
  • the method according to the invention thus contributes to the improvement of technical and economic aspects of the manufacturing of L-lysine or L-lysine containing products.
  • the bacterium provided in the method according to the invention contains in its chromosome a polynucleotide encoding an amino acid sequence of a polypeptide comprising the nucleotide sequence of positions 40 to 1923 of SEQ ID NO: 1 the nucleobases from position 1069 to 1071 being gtt, gtc, gta or gtg, preferably gtc.
  • nucleotide sequence of positions 40 to 1923 of SEQ ID NO: 1 the nucleobase at position 1070 being thymine (t).
  • nucleotide sequence of positions 40 to 1923 of SEQ ID NO:1 is identical to the nucleotide sequence of positions 40 to 1923 of SEQ ID NO:3.
  • the bacterium provided in the method according to the invention contains in its chromosome a polynucleotide encoding an amino acid sequence of a polypeptide comprising the nucleotide sequence of positions 40 to 1926 of SEQ ID NO: 1 the nucleobases from position 1069 to 1071 being gtt, gtc, gta or gtg, preferably gtc.
  • nucleotide sequence of positions 40 to 1926 of SEQ ID NO: 1 the nucleobase at position 1070 being thymine (t).
  • nucleotide sequence of positions 40 to 1926 of SEQ ID NO: 1 is identical to the nucleotide sequence of positions 40 to 1926 of SEQ ID NO:3.
  • the bacterium provided in the method according to the invention contains in its chromosome a polynucleotide encoding an amino acid sequence of a polypeptide comprising the nucleotide sequence of SEQ ID NO: 1 the nucleobases from position 1069 to 1071 being gtt, gtc, gta or gtg, preferably gtc.
  • nucleotide sequence of SEQ ID NO: 1 the nucleobase at position 1070 being thymine (t).
  • nucleotide sequence of SEQ ID NO: 1 the nucleotides from positions 1069 to 1071 being gtc is identical to the nucleotide sequence of SEQ ID NO:3.
  • L-lysine where mentioned herein, in particular in the context of product formation, also comprises their ionic forms and salts, for example L-lysine mono hydrochloride or L-lysine sulfate.
  • bacteria of the species Corynebacterium glutamicum are used.
  • Suitable bacteria for the method of this invention are L-lysine excreting strains of Corynebacterium glutamicum , for example L-lysine excreting strains obtained by one or several steps of strain development from strain ATCC13032 and the like and modified as described in this invention.
  • Strain ATCC13032 (also available as DSM20300) is the taxonomic type strain of the species Corynebacterium glutamicum .
  • a taxonomic study of this group of bacteria based on DNA-DNA hybridization was done by Liebl et al. (International Journal of Systematic Bacteriology 41(2), 255-260, 1991).
  • a comparative analysis of various strains of the species Corynebacterium glutamicum based on genome sequence analysis was provided by Yang and Yang (BMC Genomics 18(1):940).
  • L-lysine excreting strains of the species Corynebacterium glutamicum are widely known in the art and can be modified as described in the present invention.
  • U.S. Pat. No. 7,338,790 B2 describes strain DM1797. It is deposited at the DSMZ (Braunschweig, Germany) under accession number DSM16833.
  • DM1797 is an aminoethylcystein resistant mutant of strain ATCC13032 obtained after N′-methyl-N-nitro-nitrosoguanidine mutagenesis.
  • Blombach et al. Applied and Environmental Microbiology 75(2), 419-427, 2009
  • describe strain DM1933 which is deposited under accession number DSM25442.
  • Strain DM1933 was obtained from ATCC13032 by several steps of strain development. Furthermore L-lysine excreting Corynebacterium glutamicum strain DM2031, deposited at the DSMZ (Braunschweig, Germany) under the accession number DSM32514 may be used. Strain DM2031 is a further developed derivative of DM1933 having enhanced L-lysine excretion ability. Other L-lysine excreting Corynebacterium glutamicum strains are e.g. described in WO2008033001 A1 and EP0841395 A1.
  • L-lysine excreting strains of the species Corynebacterium glutamicum typically contain a polynucleotide coding for a feedback resistant aspartate kinase polypeptide variant.
  • a feedback resistant aspartate kinase polypeptide variant means an aspartate kinase which is less sensitive, or desensitized, to inhibition by mixtures of L-lysine and L-threonine, e.g. 10 mM each, or mixtures of the L-lysine analogue S-(2-aminoethyl)-L-cysteine and L-threonine, e.g.
  • said L-lysine excreting strains of the species Corynebacterium glutamicum modified as described in the present invention preferably contain at least one copy of a polynucleotide coding for a feedback resistant aspartate kinase polypeptide variant desensitized to inhibition by mixtures of L-lysine and L-threonine.
  • Said polynucleotide encoding said aspartate kinase polypeptide variant can be expressed by its natural promoter, i.e. the promoter contained in strain ATCC13032, or any other suitable promoter known in the art.
  • SEQ ID NO:5 shows the nucleotide sequence of the coding sequence of the aspartate kinase polypeptide of strain ATCC13032 and SEQ ID NO:6 the amino acid sequence of the encoded polypeptide. It is known in the art (see U.S. Pat. No. 6,893,848) that exchange of the amino acid Thr at position 311 of SEQ ID NO:6 for Ile imparts the enzyme feedback resistance to inhibition by mixtures of L-lysine and L-threonine.
  • amino acid sequence of said feedback resistant aspartate kinase polypeptide comprises the amino acid sequence of SEQ ID NO:6 containing isoleucine instead of threonine at position 311.
  • Said amino exchange can be achieved by exchanging the nucleobase cytosine (c) at position 932 of SEQ ID NO:5 to give thymine (t).
  • the acc codon for threonine is thus altered to the atc codon for isoleucine.
  • sequence coding for a feedback resistant aspartate kinase polypeptide begins with an atg start codon.
  • DSM denotes the depository Deutsche Sammlung fiir Mikroorganismen und Zellkulturen (DSMZ) located in Braunschweig, Germany.
  • ATCC denotes the depository American Type Culture Collection located in Manassas, Va. US.
  • Corynebacterium glutamicum in particular strain ATCC13032 and L-lysine excreting strains obtained therefrom during a strain development program, contain in their chromosome a, in particular one, gene encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:2.
  • the coding sequence may contain silent mutations which do not alter the amino acid sequence of the polypeptide. This context is also known as degeneracy of the genetic code in the art.
  • a mutant bacterium according to the invention can be obtained by classical in vivo mutagenesis executed with cell populations of strains of Corynebacterium glutamicum using mutagenic substances, e.g. N-methyl-N′-nitro-N-nitrosoguanidine, or ultra violet light.
  • mutagenic substances e.g. N-methyl-N′-nitro-N-nitrosoguanidine, or ultra violet light.
  • the nucleotide sequence comprising the site of mutagenesis within the gene can be amplified by PCR using primers selected from SEQ ID NO: 1 or SEQ ID NO:3. By sequencing the PCR product the desired mutants are identified. Details concerning this approach can be found inter alia in U.S. Pat. No. 7,754,446. Real-time PCR in combination with FRET hybridization probes may also be used for mutation detection.
  • FRET is the abbreviation for fluorescence resonance energy transfer. Cyril D S Mamotte (The Clinical to Biochemist Reviews 27, 63-75 (2006)) reviews the identification of single nucleotide substitutions using this method.
  • Peters-Wendisch et al. (Microbiology 144, 915-927 (1998)) used the gene replacement method to inactivate the pyc gene of Corynebacterium glutamicum encoding pyruvate carboxylase.
  • the method was applied to the zwf gene to realize an amino acid exchange at position 321 of the amino acid sequence of the Zwf sub-unit of the glucose 6-phosphate dehydrogenase.
  • U.S. Pat. No. 7,754,446 the method was applied to the rel gene to realize an amino acid exchange at position 38 of the amino acid sequence of the GTP-pyrophosphate kinase polypeptide.
  • a mutation for example, a deletion, insertion or substitution of at least one nucleobase, is provided by an isolated polynucleotide comprising the nucleotide sequence of the gene in question or a part thereof containing the mutation.
  • nucleotide sequence of the gene in question is the gene identified by NCgl0682 also known as kup gene in the art.
  • the mutation is a substitution of at least one nucleobase located in the codon specifying the amino acid glycine at position 344 of the encoded amino acid sequence (see SEQ ID NO: 1 and SEQ ID NO:2) of the polypeptide.
  • the codon specifies a proteinogenic amino acid different from glycine, preferably leucine, isoleucine or valine, particularly preferred valine.
  • the codons specifying valine are gtt, gtc, gta and gtg.
  • the codon gtc is preferred.
  • the codon for the amino acid at position 344 has the position from 1069 to 1071 in SEQ ID NO: 1 or SEQ ID NO:3.
  • the nucleotide sequence from position 1069 to 1071, in particular the nucleotide at position 1070, may also be referred to as site of mutation.
  • the mutated nucleotide sequence of the gene in question or a part thereof containing the mutation comprises i) a nucleotide sequence at the 5′-end of the site of mutation, which is also referred to as 5′-flanking sequence or upstream sequence in the art, ii) a nucleotide sequence at the 3′-end of the site of mutation, which is also referred to as 3′-flanking sequence or downstream sequence in the art, and iii) the nucleotide sequence of the site of mutation between i) and ii).
  • Said 5′-flanking sequence and 3′-flanking sequence required for homologous recombination typically have a length of at least 200 bp, at least 400 bp, at least 600 bp or at least 800 bp.
  • the maximum length typically is 1000 bp, 1500 bp or 2000 bp.
  • SEQ ID NO:7 An example of a polynucleotide comprising a mutated nucleotide sequence in the context of the present invention is shown in SEQ ID NO:7.
  • the nucleotide sequence of SEQ ID NO:7 from positions 9 to 1724 corresponds to SEQ ID NO:3 from positions 213 to 1928.
  • the polynucleotide shown in SEQ ID NO:7 contains at its 5′- and 3′-end recognition sites for the restriction endonuclease XbaI useful for cloning purposes.
  • SEQ ID NO:7 contains part of the coding sequence of the variant of the NCgl0682 polypeptide described in this invention.
  • the 5′-flanking sequence consists of the nucleotide sequence from positions 9 to 865 of SEQ ID NO:7.
  • the 3′-flanking sequence consists of the nucleotide sequence from positions 867 to 1724 of SEQ ID NO:7.
  • the site of mutation is at position 866 of SEQ ID NO:7.
  • the mutated nucleotide sequence provided is cloned into a plasmid vector, e.g. pK18mobsacB described by Schafer et al. (Gene 145, 69-73 (1994)), that is not capable of autonomous replication in Corynebacterium glutamicum .
  • Said plasmid vector comprising said mutated nucleotide sequence is subsequently transferred into the desired strain of Corynebacterium glutamicum by transformation using electroporation or conjugation.
  • the mutation is incorporated in the Corynebacterium glutamicum chromosome.
  • the nucleotide sequence of the gene in question contained in the chromosome of said desired strain is replaced by the mutated nucleotide sequence.
  • the presence of the mutation in the desired strain is then confirmed e.g. by analysis of the nucleotide sequence or real-time PCR using FRET as described above.
  • An event of homologous recombination may also be referred to as crossing over.
  • the L-lysine excreting Corynebacterium glutamicum strains provided for the method of the present invention have the ability to excrete ⁇ 0.25 g/l, preferably ⁇ 0.5 g/l, particularly preferred ⁇ 1.0 g/l, very particularly preferred ⁇ 2.0 g/l of L-lysine in a suitable medium under suitable conditions.
  • a Corynebacterium glutamicum modified in accordance with the present invention and having the ability to excrete L-lysine is cultivated in a suitable medium under suitable conditions. Due to said ability to excrete said L-lysine the concentration of the L-lysine increases and accumulates in the medium during the fermentative process and the L-lysine is thus produced.
  • a suitable medium used for the production of L-lysine by a fermentative process contains a carbon source, a nitrogen source, a phosphorus source, inorganic ions and other organic compounds as required.
  • Suitable carbon sources include glucose, fructose, sucrose as well as the corresponding raw materials like starch hydrolysate, molasses or high fructose corn syrup.
  • organic nitrogen-containing compounds such as peptones, meat extract, soy bean hydrolysates or urea
  • inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate, ammonium gas or aqueous ammonia can be used.
  • phosphorus source phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used.
  • Inorganic ions like potassium, sodium, magnesium, calcium, iron and further trace elements etc. are supplied as salts of sulfuric acid, phosphoric acid or hydrochloric acid.
  • organic compounds mean essential growth factors like vitamins e. g. thiamine or biotin or L-amino acids e.g. L-homoserine.
  • the media components may be added to the culture in form of a single batch or be fed in during the cultivation in a suitable manner.
  • the pH of the culture can be controlled by employing basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds such as phosphoric acid or sulphuric acid in a suitable manner.
  • the pH is generally adjusted to a value of from 6.0 to 8.5, preferably 6.5 to 8.0.
  • antifoam agents such as, for example, fatty acid polyglycol esters.
  • suitable selective substances such as, for example, antibiotics.
  • the fermentative process is preferably carried out under aerobic conditions.
  • oxygen or oxygen-containing gas mixtures such as, for example air are introduced into the culture.
  • the fermentative process is carried out, where appropriate, at elevated pressure, for example at an elevated pressure of 0.03 to 0.2 MPa.
  • the temperature of the culture is normally from 25° C. to 40° C., preferably from 30° C. to 37° C.
  • the cultivation is continued until an amount of the L-lysine sufficient for being recovered has been formed.
  • the cultivation is then completed. This aim is normally achieved within 10 hours to 160 hours. In continuous processes, longer cultivation times are possible.
  • the fermentative process results in a fermentation broth which contains the desired to L-lysine.
  • a product containing the L-lysine is then recovered or manufactured in liquid or solid from the fermentation broth.
  • a “fermentation broth” means a medium in which a Corynebacterium glutamicum described in the invention has been cultivated for a certain time and under certain conditions.
  • the resulting fermentation broth accordingly comprises:
  • the organic by-products include compounds which may be formed by the Corynebacterium glutamicum during the fermentative process according to the present invention in addition to production of the L-lysine.
  • the fermentation broth is removed from the culture vessel or fermentation tank, collected where appropriate, and used for providing a product containing the L-lysine, in liquid or solid form.
  • the expression “recovering the L-lysine-containing product” is also used for this.
  • the L-lysine-containing fermentation broth itself which has been removed from the fermentation tank, constitutes the recovered product.
  • the fermentation broth can subsequently be subjected to extracting or substantially eliminating water from said fermentation broth.
  • water In particular at least 40% (w/w), preferred at least 90% (w/w), more preferred at least 95% (w/w) water are extracted from the to fermentation broth.
  • Removal of the biomass can be achieved inter alia by centrifugation, filtration or decantation or a combination thereof.
  • Manufacturing of an L-lysine product may also comprise a purification step. preferably selected from the group consisting ion exchange chromatography, treatment with activated carbon or crystallization.
  • a product containing L-lysine ⁇ HCl preferably containing ⁇ 80% L-lysine ⁇ HCl, particularly preferred ⁇ 90% L-lysine ⁇ HCl or ⁇ 95% L-lysine ⁇ HCl can be obtained.
  • L-lysine to determine its concentration at one or more time(s) during the fermentation can take place by separating the L-lysine by means of ion exchange chromatography, preferably cation exchange chromatography, with subsequent post-column derivatization using ninhydrin, as described in Spackman et al. (Analytical Chemistry 30: 1190-1206 (1958)). It is also possible to employ ortho-phthalaldehyde rather than ninhydrin for post-column derivatization. An overview article on ion exchange chromatography can be found in Pickering (LC.GC (Magazine of Chromatographic Science 7(6):484-487 (1989)).
  • RP reversed-phase chromatography
  • HPLC high-performance liquid chromatography
  • Kanamycin Kanamycin solution from Streptomyces kanamyceticus from Sigma Aldrich (St. Louis, USA, Cat. no. K0254).
  • Nalidixic acid Nalidixic acid sodium salt from Sigma Aldrich (St. Louis, USA, Cat. no. N4382).
  • a. LB broth from Merck (Darmstadt, Germany; Cat. no. 110285) was used to cultivate E. coli strains in liquid medium.
  • the liquid cultures (10 ml liquid medium per 100 ml Erlenmeyer flask with 3 baffles) were incubated in the Infors HT Multitron standard incubator shaker from Infors GmbH (Einsbach, Germany) at 37° C. and 200 rpm.
  • LB agar from Merck (Darmstadt, Germany Cat. no. 110283) was used for cultivation of E. coli strains on agar plates. The agar plates were incubated at 37° C. in an INCU-Line® mini incubator from VWR (Radnor, USA).
  • BHI Brain heart infusion broth
  • Merck Merck (Darmstadt, Germany; Cat. no. 110493) was used to cultivate C. glutamicum strains in liquid medium.
  • the liquid cultures (10 ml liquid medium per 100 ml Erlenmeyer flask with 3 baffles) were incubated in the Infors HT Multitron standard incubator shaker from Infors GmbH (Einsbach, Germany) at 33° C. and 200 rpm.
  • Brain heart agar (BHI-agar) from Merck (Darmstadt, Germany; Cat. no. 113825) was used for cultivation of C. glutamicum strains on agar plates. The agar plates were incubated at 33° C. in an incubator from Heraeus Instruments with Kelvitron® temperature controller (Hanau, Germany).
  • the optical density of bacterial suspensions in shake flask cultures was determined at 600 nm (OD600) using the BioPhotometer from Eppendorf AG (Hamburg, Germany).
  • the optical density of bacterial suspensions produced in the Wouter Duetz (WDS) micro fermentation system was determined at 660 nm (OD660) with the GENiosTM plate reader from Tecan Group AG (Mannedorf, Switzerland).
  • Bacterial suspensions with a maximum volume of 50 ml were caused to sediment using 15 ml or 50 ml centrifuge tubes (e.g. FalconTM 50 ml Conical Centrifuge Tubes) using an Eppendorf 5810 R centrifuge for 10 min. at 4.000 rpm.
  • 15 ml or 50 ml centrifuge tubes e.g. FalconTM 50 ml Conical Centrifuge Tubes
  • Eppendorf 5810 R centrifuge for 10 min. at 4.000 rpm.
  • FRET fluorescence resonance energy transfer
  • the FRET hybridization donor probe was labelled with the fluorescent dye fluorescein and the acceptor probe with the fluorescent dye LC-Red640.
  • the detection method comprised three steps: colony PCR, probe hybridization and subsequent melting curve analysis. The method is simply referred to as real-time PCR herewith.
  • the oligonucleotides used were synthesized by eurofins genomics GmbH (Ebersberg, Germany).
  • PCR template As PCR template the total DNA contained in a colony was used. It was prepared by taking cell material with a toothpick from a colony on an agar plate and placing the cell material to directly into the PCR reaction tube. The cell material was heated for 10 sec. with 800 W in a microwave oven type Mikrowave & Grill from SEVERIN Elektrogerate GmbH (Sundem, Germany) and then the PCR reagents were added to the template in the PCR reaction tube.
  • the Type-it® Fast SNP probe PCR Kit (Type-it Kit) from Qiagen (Hilden, Germany, Cat.No. 206045) was used for real-time detection of the mutations. Therefore 2.5 ⁇ l of the Qiagen Fast SNP Puffer (2 ⁇ ) was mixed with 0.5 ⁇ l of each of the LC-PCR-Primers [10 ⁇ M] and 0.5 ⁇ l of each of the 1:500 diluted acceptor and donor probe [100 pmol/ ⁇ l] to get the mastermix for the real-time PCR.
  • E. coli K-12 strain S17-1 was used as donor for conjugational transfer of plasmids based on pK18mobsacB from E. coli to C. glutamicum .
  • Strain S17-1 is described by Simon. R. et al. (Bio/Technology 1, 784-794, 1983). It is available from the American Type Culture Collection under the access number ATCC47055.
  • Chemically competent E. coli S 17-1 cells were made as follows: A preculture of 10 ml LB medium (10 ml liquid medium per 100 ml Erlenmeyer flask with 3 baffles) was inoculated with 100 ⁇ l bacterial suspension of strain S17-1 and the culture was incubated overnight for about 18 h at 37° C. and 250 rpm. The main culture (70 ml LB contained in a 250 ml Erlenmeyer flask with 3 baffles) was inoculated with 300 ⁇ l of the preculture and incubated up to an OD600 of 0.5-0.8 at 37° C. The culture was centrifuged for 6 min. at 4° C. and 4000 rpm and the supernatant were discarded.
  • the cell pellet was resuspended in 20 ml sterile, ice-cold 50 mM CaCl 2 solution and incubated on ice for 30 min. After another centrifugation step, the pellet was resuspended in 5 ml ice-cold 50 mM CaCl 2 solution and the suspension incubated on ice for 30 min. The cell suspension was then adjusted to a final concentration of 20% glycerol (v/v) with 85% (v/v) sterile ice-cold glycerol. The suspension was divided into 50 ⁇ l aliquots and stored at ⁇ 80° C. To transform S17-1 cells, the protocol according to Tang et al. (Nucleic Acids Res. 22(14), 2857-2858, 1994) with a heat shock of 45 sec. was used.
  • the pK18mobsacB plasmid system described by Schafer et al. was used to integrate desired DNA fragments into the chromosome of C. glutamicum .
  • a modified conjugation method of Schafer et al. was used to transfer the respective plasmid into the desired C. glutamicum recipient strain.
  • Sterile toothpicks were used to transfer the transconjugants onto BHI agar, which was supplemented with 25 mg/l kanamycin and 50 mg/l nalidixic acid.
  • the agar plates were incubated for 20 h at 33° C.
  • the cultures of the respective transconjugants produced in this manner were then propagated further for 24 h at 33° C. in 10 ml BHI medium contained in 100 ml Erlenmeyer flasks with 3 baffles.
  • An aliquot was taken from the liquid culture suitably diluted and plated (typically 100 to 200 ⁇ l) on BHI agar which was supplemented with 10% sucrose.
  • the agar plates were incubated for 48 h at 33° C.
  • the colonies growing on the sucrose containing agar plates were then examined for the phenotype kanamycin sensitivity. To do so a toothpick was used to remove cell material from the colony and to transfer it onto BHI agar containing 25 mg/l kanamycin and onto BHI agar containing 10% sucrose. The agar plates were incubated for 60 h at 33° C. Clones that proved to be sensitive to kanamycin and resistant to sucrose were examined for integration of the desired DNA fragment by means of real-time PCR.
  • E. coli - and C. glutamicum strains glycerol stocks were prepared. Selected E. coli clones were cultivated in 10 ml LB medium supplemented with 2 g/l glucose. Selected C. glutamicum clones were cultivated in twofold concentrated BHI medium supplemented with 2 g/l glucose. Cultures of plasmid containing E. coli strains were supplemented with 50 mg/l kanamycin. Cultures of plasmid containing C. glutamicum strains were supplemented with 25 mg/l kanamycin. The medium was contained in 100 ml Erlenmeyer flasks with 3 baffles.
  • Precultures of the strains were done in 10 ml twofold concentrated BHI medium.
  • the medium was contained in a 100 ml Erlenmeyer flask with 3 baffles. It was inoculated with 100 ⁇ l of a glycerol stock culture and the culture incubated for 24 h at 33° C. and 200 rpm.
  • the main cultures were done by inoculating the 2.5 ml medium containing wells of the 24 Well WDS-Plate with an aliquot of the preculture to give an optical density OD600 of 0.1.
  • CGXII medium As medium for the main culture CGXII medium was used.
  • the composition of the CGXII medium is shown in table 3.
  • the glucose concentration in the suspension was analysed with the blood glucose-meter to OneTouch Vita® from LifeScan (Johnson & Johnson Medical GmbH, Neuss, Germany). After cultivation the culture suspensions were transferred to a deep well microplate. A part of the culture suspension was suitably diluted to measure the OD600. Another part of the culture was centrifuged and the concentration of L-amino acids, in particular L-lysine, and residual glucose were analysed in the supernatant.
  • the concentration of L-lysine and other L-amino acids, e.g. L-valine, in the culture supematants was determined by ion exchange chromatography using a SYKAM S433 amino acid analyser from SYKAMmaschines GmbH (Firstenfeldbruck, Germany). As solid phase a column with spherical, polystyrene-based cation exchanger (Peek LCA N04/Na, dimension 150 ⁇ 4.6 mm) from SYKAM was used. Depending on the L-amino acid the separation takes place in an isocratic run using a mixture of buffers A and B for elution or by gradient elution using said buffers.
  • buffer A an aqueous solution containing in 201 263 g trisodium citrate, 120 g citric acid, 1100 ml methanol, 100 ml 37% HCl and 2 ml octanoic acid (final pH 3.5) was used.
  • buffer B an aqueous solution containing in 20 l 392 g trisodium citrate, 100 g boric acid and 2 ml octanoic acid (final pH 10.2) was used.
  • the free amino acids were coloured with ninhydrin through post-column derivatization and detected photometrically at 570 nm.
  • a SANplus multi-channel continuous flow analyser from SKALAR analytic GmbH (Erkelenz, Germany) was used to determine the concentration of glucose in the supernatant.
  • Glucose was detected with a coupled-enzyme assay (Hexokinase/Glucose-6-Phosphate-Dehydrogenase) via NADH formation.
  • Strain DM1933 is an L-lysine producer described by Blombach et al. (Applied and Environmental Microbiology 75(2), 419-427, 2009). It can be obtained at the DSMZ under accession number DSM25442.
  • the nucleotide sequence of the chromosome of strain DM1933 was determined by Illumina whole-genome sequencing technology (Illumina Inc., San Diego, Calif., US). See e.g. Benjak et al. (2015) Whole-Genome Sequencing for Comparative Genomics and De Novo Genome Assembly. In: Parish T., Roberts D. (eds) Mycobacteria Protocols. Methods in Molecular Biology, Vol 1285. Humana Press, NY, US) and Bennet, S. (Pharmacogenomics 5(4), 433-438, 2004).
  • nucleotide sequence of the kup coding sequence (locus_tag NCgl0682) of strain DM1933 including the nucleotide sequence upstream and downstream thereof is identical to that of ATCC13032 shown in SEQ ID NO: 1.
  • DM1933 contains in its chromosome a variant of the aspartokinase gene encoding a feedback resistant aspartokinase polypeptide.
  • Said feedback resistant aspartokinase polypeptide has the amino acid sequence of SEQ ID NO:6 of the sequence listing, wherein the amino acid threonine (Thr) at position 311 of the amino acid sequence is replaced by isoleucine (Ile).
  • Thr amino acid threonine
  • Ile isoleucine
  • Plasmid pK18mobsacB_kup_G344V was constructed to enable incorporation of the mutation causing the amino acid exchange G344V into the nucleotide sequence of the kup coding sequence of strain DM1933.
  • the plasmid is based on the mobilizable vector pK18mobsacB described by Schafer et al. (Gene 145, 69-73, 1994).
  • kup_G344V polynucleotide according to SEQ ID NO:7 was synthesized and subcloned into pK18mobsacB by GeneArt (ThermoFisher Scientific (Waltham, USA)).
  • the two polynucleotides i.e. the vector pK18mobsacB and the polynucleotide kup_G344V were both treated with XbaI, ligated and the ligation mixture used to transform E. coli.
  • DNA of plasmid pK18mobsacB_kup_G344V was isolated from a transformant.
  • the plasmid pK18mobsacB_kup_G344V obtained in example 2 was used to incorporate the mutation (see nucleotide position 1070 of SEQ ID NO: I and SEQ ID NO:3 and nucleotide position 866 of SEQ ID NO:7) leading to the amino acid exchange G344V (see nucleotide positions 1069-1071 of SEQ ID NO:1 and SEQ ID NO:3, amino acid position 344 of SEQ ID NO:2 and SEQ ID NO:4 and nucleotide positions 865-867 of SEQ ID NO:7) into the chromosome of the L-lysine producer DM1933.
  • Transconjugant clones were analyzed by real-time PCR using the Type-it Kit and the primers LC-Ncgl0682_1 and LC-Ncgl0682_2 for PCR amplification and NCgl0682_344_C as acceptor probe and NCgl0682_G344V_A as donor probe for melting curve analysis (table 4).
  • transconjugant clones thus characterized was called DM1933_kup_G344V.
  • a glycerol stock culture of the transconjugant clone was prepared and used as starting material for further investigations.
  • Strains DM1933 (reference) and DM1933_kup_G344V obtained in example 3 were analyzed for their ability to produce L-lysine from glucose by batch cultivation using the cultivation system according to Wouter Duetz.
  • strain DM1933_kup_G344V TABLE 5 L-lysine production by strain DM1933_kup_G344V.
  • the experiment shows that L-lysine production was increased in strain DM1933_kup_G344V as compared to the parent strain DM1933.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
US16/701,634 2018-12-18 2019-12-03 Method for the fermentative production of L-lysine using C. glutamicum strains with a mutated kup transporter Active US10717999B2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP18213573.1 2018-12-18
EP18213573 2018-12-18
EP18213573.1A EP3670525A1 (de) 2018-12-18 2018-12-18 Verfahren zur fermentativen herstellung von l-lysin unter verwendung von c.-glutamicum-stämmen mit einem mutierten kup-transporter

Publications (2)

Publication Number Publication Date
US20200190546A1 US20200190546A1 (en) 2020-06-18
US10717999B2 true US10717999B2 (en) 2020-07-21

Family

ID=65009522

Family Applications (1)

Application Number Title Priority Date Filing Date
US16/701,634 Active US10717999B2 (en) 2018-12-18 2019-12-03 Method for the fermentative production of L-lysine using C. glutamicum strains with a mutated kup transporter

Country Status (5)

Country Link
US (1) US10717999B2 (de)
EP (2) EP3670525A1 (de)
CN (1) CN111334534A (de)
BR (1) BR102019026898A2 (de)
RU (1) RU2019139788A (de)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113563436B (zh) * 2021-08-23 2024-06-25 宁夏伊品生物科技股份有限公司 Yh66-rs03880基因改造得到的工程菌及其在制备缬氨酸中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001000805A2 (en) 1999-06-25 2001-01-04 Basf Aktiengesellschaft Corynebacterium glutamicum genes encoding proteins involved in membrane synthesis and membrane transport
WO2009141330A1 (de) 2008-05-20 2009-11-26 Evonik Degussa Gmbh Verfahren zur herstellung von l-aminosäuren

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3473042B2 (ja) 1992-04-28 2003-12-02 味の素株式会社 変異型アスパルトキナーゼ遺伝子
AU705550B2 (en) 1995-06-07 1999-05-27 Ajinomoto Co., Inc. Method of producing L-lysine
JP4526710B2 (ja) 1999-04-19 2010-08-18 協和発酵バイオ株式会社 新規な脱感作型アスパルトキナーゼ
JP4623825B2 (ja) 1999-12-16 2011-02-02 協和発酵バイオ株式会社 新規ポリヌクレオチド
US6844176B1 (en) 2001-10-16 2005-01-18 Degussa Ag Alleles of the lysC gene from corynebacteria
DE102005032426A1 (de) 2004-12-18 2006-06-22 Degussa Ag Allele des gnd-Gens aus coryneformen Bakterien
US20070092951A1 (en) 2005-03-24 2007-04-26 Degussa Ag Alleles of the zwf gene from coryneform bacteria
AU2007295159B2 (en) 2006-09-15 2012-09-13 Cj Cheiljedang Corporation A Corynebacteria having enhanced L-lysine productivity and a method of producing L-lysine using the same
DE102006048882A1 (de) 2006-10-17 2008-04-24 Evonik Degussa Gmbh Allele des rel-Gens aus coryneformen Bakterien
CN105483145B (zh) 2011-12-21 2021-04-06 Cj第一制糖株式会社 利用具有产生l-赖氨酸能力的微生物产生l-赖氨酸的方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001000805A2 (en) 1999-06-25 2001-01-04 Basf Aktiengesellschaft Corynebacterium glutamicum genes encoding proteins involved in membrane synthesis and membrane transport
WO2009141330A1 (de) 2008-05-20 2009-11-26 Evonik Degussa Gmbh Verfahren zur herstellung von l-aminosäuren
US20090311758A1 (en) 2008-05-20 2009-12-17 Evonik Degussa Gmbh Process for preparing l-amino acids
US8697850B2 (en) 2008-05-20 2014-04-15 Evonik Degussa Gmbh Process for preparing L-amino acids
US20140127787A1 (en) 2008-05-20 2014-05-08 Evonik Degussa Gmbh Process for preparing l-amino acids
US9422568B2 (en) 2008-05-20 2016-08-23 Evonik Degussa Gmbh Process for preparing L-amino acids

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
European Search Report dated Jun. 17, 2019 in European Application 18213573.1.
Extended Search Report dated May 4, 2020 in European Application No. 19215284.1, 6 pages.
Follman et al., Journal of Bacteriology, vol. 191, No. 9, Mar. 6, 2009, pp. 2944-2952.

Also Published As

Publication number Publication date
EP3670526A1 (de) 2020-06-24
CN111334534A (zh) 2020-06-26
RU2019139788A (ru) 2021-06-07
EP3670526B1 (de) 2021-09-01
US20200190546A1 (en) 2020-06-18
BR102019026898A2 (pt) 2020-10-13
EP3670525A1 (de) 2020-06-24

Similar Documents

Publication Publication Date Title
EP3467114A1 (de) Verfahren zur fermentativen herstellung von l-aminosäuren
EP3456834B1 (de) Verfahren zur fermentativen herstellung von l-aminosäuren
EP3498854B1 (de) Verfahren zur fermentativen herstellung von l-lysin
EP3628742B1 (de) Verfahren zur fermentativen herstellung von l-lysin
US10717999B2 (en) Method for the fermentative production of L-lysine using C. glutamicum strains with a mutated kup transporter
US10829746B2 (en) Method for the fermentative production of L-lysine
EP3599282B1 (de) Verfahren zur fermentativen herstellung von l-lysin
US11198895B2 (en) Method for the fermentative production of L-lysine using an L-lysine excreting bacterium of the species Corynebacterium glutamicum having a completely or partly deleted WHIB4 gene
EP3594355A1 (de) Verfahren zur fermentativen herstellung von l-lysin
WO2021048353A1 (en) Coryneform bacteria with a heterologous threonine transporter and their use in the production of l-threonine
US20240271169A1 (en) Method for the fermentative production of l-lysine using c. glutamicum strains expressing modified gluconate repressor proteins gntr1 and gntr2

Legal Events

Date Code Title Description
FEPP Fee payment procedure

Free format text: ENTITY STATUS SET TO UNDISCOUNTED (ORIGINAL EVENT CODE: BIG.); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

AS Assignment

Owner name: EVONIK OPERATIONS GMBH, GERMANY

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:BEKEL, THOMAS;VOSS, KORNELIA;THIERBACH, GEORG;AND OTHERS;SIGNING DATES FROM 20191213 TO 20200121;REEL/FRAME:051631/0939

STCF Information on status: patent grant

Free format text: PATENTED CASE

MAFP Maintenance fee payment

Free format text: PAYMENT OF MAINTENANCE FEE, 4TH YEAR, LARGE ENTITY (ORIGINAL EVENT CODE: M1551); ENTITY STATUS OF PATENT OWNER: LARGE ENTITY

Year of fee payment: 4